This invention relates to novel drug delivery compositions which provide for the improved uptake of therapeutic agents across mucosal surfaces.
Polar drugs, including high molecular weight peptides, proteins and polysaccharides, are typically not effectively absorbed across mucosal membranes, such as the gastrointestinal tract, the eye, the vagina, the nasal cavity or the rectum. Such molecules are thus normally only given by injection, which inevitably gives rise to well known problems associated with patient compliance, the cost of treatment, as well as the potentially harmful effects, such as phlebitis and pain, of the injection.
It is well known in the literature that the absorption of polar molecules across mucosal membranes may be greatly improved if they are administered in combination with so-called xe2x80x9cabsorption enhancersxe2x80x9d. Examples of absorption enhancers which have been described in the literature include non-ionic surfactants, cyclodextrins, pholspholipids and bile salts. (For a review see Davis et al (eds.), Delivery Systems for Peptide Drugs, Plenum Press, New York, 1987; and Lee (ed.), Peptide and Protein Delivery, Marcel Dekker Inc., New York, 1991.)
EP-A-023 359 and EP-A-122 023 describe powdery pharmaceutical compositions for application to the nasal mucosa, as well as methods for the administration of such compositions. The pharmaceutical compositions allow polypeptides and derivatives thereof to be effectively absorbed through the nasal mucosa. Similarly, U.S. Pat. No. 4,226,849 describes a method for administering a powdery medicament to the nasal mucosa, in which the preferred composition has mucoadhesive properties.
Formulations based on microspheres for mucosal delivery have been described in WO 88/09163. The formulations contain certain enhancers to aid effective penetration of the mucosa by the drug. WO 89/03207 describes microsphere formulations which do not require an enhancer.
Chitosan is a derivative of chitin or poly-N-acetyl-D-glucosamine in which the greater proportion of the N-acetyl groups have been removed through hydrolysis. It is available from several suppliers including Pronova, Drammen, Norway, and, depending on the grade selected, is soluble in water and/or aqueous acid up to pH values of between 6.0 and 7.0.
Chitosan has previously been used to precipitate proteinaceous material and to make surgical sutures. It has also been employed previously in oral drug formulations in order to improve the dissolution of poorly soluble drugs (see Sawavanagi et al, Chem. Pharm. Bull., 31 (1983) 2062-2068) or for the sustained release of drugs by a process of slow erosion from a hydrated compressed matrix (Nagai et al, Proc. Jt. US Jpn. Semin. Adv. Chitin Chitosan Relat. Enzymes, 21-39, Zikakis J. P. (ed.), Academic Press, Orlando, 1984).
WO 90/09780 describes a composition comprising a drug and a polycationic substance (e.g. chitosan) that promotes the transport of the drug across mucosal membranes. The composition may also comprise microspheres of the polycationic substance.
WO 96/05810 describes a composition comprising a pharmacologically active compound and particles, preferably powders or microspheres, of chitosan or a chitosan derivative or salt, where the particles are either solidified or partially cross-linked such that they have a zeta-potential of between +0.5 and +50 mV. Solidified particles are made by treating particles made from a water soluble chitosan salt with an alkaline agent, such as sodium hydroxide, in non-acid containing water to render them insoluble.
Chitosan microspheres have also been produced for use in enhanced chromatographic separation (Li Q. et al, Biomater. Artif. Cells Immobilization Biotechnology, 21 (1993) 391-398), for the topical delivery of drugs (Machida Y., Yakugaku Zasshl., 113 (1993) 356-368), for drug targeting after injection (Ohya Y et al, J. Microencap., 10 (1993) 1-9), as an implantable controlled release delivery system (Jameela and Jayakrishnan, Biomaterials, 16 (1995) 769-775) and for the controlled release of drugs (see Bodmeier R. et al, Pharm. Res., 6 (1989) 413-417 and Chithambara et al, J. Pharm. Pharmacol., 44 1992, 283-286).
EP 454044 and EP 486959 describe polyelectrolyte microparticles or polysaccharide microspheres, including chitosan microspheres, for use in the controlled release of drugs. Chitosan microspheres crosslinked with glutaraldehyde have also been described in JP 539149.
Gelatin is a purified protein obtained either by partial acid hydrolysis (type A) or by partial alkaline hydrolysis (type B) of animal collagen. Type A gelatin is cationic with an isoelectric point between pH values of 7 and 9, whereas type B gelatin is anionic with an isoelectric point between pH values of 4.7 and 5. Gelatin is known to swell and soften when immersed in cold water, eventually absorbing between 5 and 10 times its own weight in water. It is soluble in hot water, forming a gel on cooling. Gelatin is used as a haemostatic in surgical procedures as an absorbable film or sponge, which can absorb many times its own weight in blood. It is also employed as a plasma substitute, and may be used in the preparation of pastes, pastilles, suppositories, tablets and hard and soft capsule shells for oral formulations.
The production of gelatin microspheres has been widely described in the literature. Gelatin microspheres have been produced by an emulsification method involving crosslinking with glutaraldehyde, producing microspheres of less than 2 xcexcm in diameter (Tabata and Ikada, Pharm. Res. 6 (1989) 422-427). Cortesi et al (Int. J. Pharm. 105 (1994) 181-186), Natruzzi et al (J. Microencapsulation, 11 (1994) 294-260) and Esposito et al (Int. J. Pharm., 117 (1995) 151-158) have reported the production of microspheres of a mean diameter of 22 xcexcm using a coacervation emulsification method. Microspheres as produced by the latter processes were not crosslinked. Microspheres of a smaller size have been produced according to a similar method by Esposito et al (Pharm. Sci. Commun. 4 (1994) 239-246). The type of gelatin (A or B) used in these studies was not specified.
The production of microspheres by complexation, between a negatively charged material such as alginate and a positively charged chitosan has been described in the literature. For example. Polk et al, J. Pharm. Sci., 83 (1994) 178-185) describes the production of clhitosan-alginate microspheres by the addition of an alginate solution to a solution of chitosan and calcium ions. The highest concentration of chitosan used in the microsphere formulations was 5.2% w/w. Similarly, the formation of complex coacervates between oppositely charged polyions, namely a positively charged chitosan and a negatively charged type B gelatin has been described by Remunan-Lopez and Bodmeier (Int. J. Pharm. 135 (1996) 63-72). These workers found the optimum chitosan:gelatin ratio to be in the range 1:10 to 1:20. The coacervate was obtained in a dry form by decanting the supernatant after centrifugation and drying at 60xc2x0 C. We have now found, surprisingly, that microparticles, produced from a combination of a chitosan and a cationic type A gelatin, possess particularly advantageous properties, which enable the improved transport of therapeutic agents, including polar drugs, across mucosal surfaces such as the nasal cavity.
Thus, according to a first aspect of the invention there is provided a composition comprising a mixture of chitosan and type A. cationic, gelatin, together with a therapeutic agent (hereinafter referred to as xe2x80x9cthe compositions according to the inventionxe2x80x9d).
By xe2x80x9cmixture of chitosan and type A gelatinxe2x80x9d we include any composition comprising a chitosan, as defined hereinafter, and a type A gelatin, as defined hereinafter, whether a physical and/or chemical association between these two constituents exists or not.
The term xe2x80x9cchitosanxe2x80x9d will be understood by those skilled in the art to include all derivatives of chitin, or poly-N-aceryl-D-glucosamine (including all polyglucosamine and oligomers of glucosamine materials of different molecular weights), in which the greater proportion of the N-acetyl groups have been removed through hydrolysis. We prefer that the chitosan has a positive charge.
Chitosan, chitosan derivatives or salts (e.g. nitrate, phosphate, sulphate, hydrochloride, glutamate, lactate or acetate salts) of chitosan may be used. We use the term chitosan derivatives to include ester, ether or other derivatives formed by bonding of acyl and/or alkyl groups with OH groups, but not the NH2 groups, of chitosan. Examples are O-alkyl ethers of chitosan and 0-acyl esters of chitosan. Modified chitosans, particularly those conjugated to polyethylene glycol, are included in this definition. Low and medium viscosity chitosans (for example CL113, G210 and CL110) may be obtained from various sources, including Pronova Biopolymer, Ltd., UK; Seigagaku America Inc., Maryland, USA; Meron (India) Pvt, Ltd., India; Vanson Ltd, Virginia, USA; and AMS Biotechnology Ltd., UK. Suitable derivatives include those which are disclosed in Roberts, Chitin Chemistry, MacMillan Press Ltd., London (1992).
The chitosan or chitosan derivative or salt used preferably has a molecular weight of 4,000 Dalton or more, preferably in the range 25,000 to 2,000,000 Dalton, and most preferably about 50,000 to 300,000 Dalton. Chitosans of different low molecular weights can be prepared by enzymatic degradation of chitosan using chitosanase or by the addition of nitrous acid. Both procedures are well known to those skilled in the art and are described in recent publications (Li et al. (1995) Plant Physiol. Biochem. 33, 599-603; Allan and Peyron, (1995) Carbohydrate Research 277, 257-272; Damard and Cartier, (1989) Int. J. Biol. Macromol. 11, 297-302).
Preferably, the chitosan is water-soluble and may be produced from chitin by deacetylation to a degree of greater than 40%, preferably between 50% and 98%, and more preferably between 70% and 90%. Particular deacetylated chitosans which may be mentioned include the xe2x80x9cSea Cure(copyright)xe2x80x9d series of chitosan glutamates available from Protan Biopolymer A/S, Drammen, Norway.
The term xe2x80x9ctype A gelatinxe2x80x9d includes all cationic proteins which are, or may be, obtained by partial acid hydrolysis of animal collagen, and excludes type B gelatins.
Although the compositions according to the invention may be prepared in a variety of physical forms using techniques which will be well known to the skilled person, we prefer that the compositions are in the form of microparticles. The term xe2x80x9cmicroparticlesxe2x80x9d includes microspheres, microcapsules and powders. However, we prefer that the microparticles are microspheres.
We have found, surprisingly, that when the compositions according to the invention are provided in the form of microparticles, such microparticles retain a positive charge and may provide for the improved transport of polar drugs across, or for the improved presentation of vaccines to muscosal surfaces, such as the nasal cavity, to such an extent that the effect is superior to that obtained for a chitosan solution, or microparticles produced from chitosan or type A gelatin alone (e.g. soluble (spray dried) chitosan microsphercs and gelatin microspheres). The effect is also similar to that obtained for partially aldehyde crosslinked chitosan microspheres, yet the compositions according to the invention are sufficiently hard/solid not to require crosslinking. We have further found that the flow properties of these chitosan/type A gelatin microparticles are superior to those of spray dried chitosan microspheres and crosslinked chitosan microspheres.
The microparticles may be prepared by spray drying, emulsification, solvent evaporation, precipitation or other methods known to a person skilled in the art. The therapeutic agent can be incorporated into the microparticles during their production or sorbed onto the microparticles after their production.
When the compositions according to the invention are in the form of microspheres, they may be prepared using for example either emulsification or spray drying techniques.
When microsphercs are prepared by spray drying, a warm mixture of chitosan and type A gelatin is spray dried with instant cooling of the resultant microspheres. The therapeutic agent may be incorporated by adsorbing onto the surface of the microspheres by freeze drying or spray drying a suspension of the microspheres with the therapeutic agent, or by physically or mechanically mixing the dried microspheres with the therapeutic agent.
However, we have found that microspheres may advantageously be prepared by warming a solution of a chitosan mixed with type A gelatin which is then emulsified and gelated by cooling. We have found that, in particular, microspheres prepared in accordance with this technique exhibit the advantageous properties referred to hereinbefore.
In the emulsification technique, the chitosan may be dissolved in water and mixed with type A gelatin under heating to 40xc2x0 C. causing the gelatin to melt. This mixture may be emulsified, at a temperature above the melting point of the gelatin, in an organic medium (e.g. a vegetable oil, such as sunflower oil, soya oil, cotton seed oil or coconut oil), in the presence of an emulsifier with a low hydrophilic-lipophilic balance (HLB) value. Such emulsifiers, which are useful for stabilising water-in-oil emulsions, are known to those skilled in the art (e.g. Span 80). The microspheres may then be solidified by decreasing the temperature of the emulsion to below 10xc2x0 C. with stirring. The microspheres may then be harvested using conventional techniques, for example by adding a pharmaceutically acceptable organic solvent, e.g. chilled acetone or petroleum ether, to the emulsion, centrifugation, washing and drying. The therapeutic agent may be incorporated into the microspheres by adding it to the chitosan/gelatin mixture before emulsification. Alternatively, the therapeutic agent may be adsorbed onto the surface of the microspheres by freeze drying or by spray drying a suspension of the microsphercs with the therapeutic agent, or by physically or mechanically mixing the dried microspheres with the therapeutic agent.
Thus, according to a further aspect of the invention there is provided a drug delivery composition in a form suitable for administration to a mucosa comprising a therapeutic agent and microparticles made from a mixture of chitosan and type A gelatin and where the agent is either incorporated into the particles during production or is adsorbed to the surface of the particles, or is present as an admixture.
Microcapsules and powders may be made by modifying the process as defined herein in accordance with techniques which are well known to those skilled in the art, or may be prepared in accordance with other techniques which will be well known to those skilled in the art, including double emulsification processes.
According to a further aspect of the invention there is provided a process for the preparation of a composition according to the invention, which process comprises preparation of type A gelatin/chitosan microparticles (i.e. microparticles comprising a mixture of type A gelatin and chitosan) by a process of spray drying or by emulsification, which emulsification may comprise warming a solution of a chitosan mixed with type A gelatin, emulsification and gelation by cooling.
The flow properties of the microparticles can be measured by methods known to those skilled in the art. One possible method involves the measurement of the Hausner Ratio where a known weight of material is poured into a measuring cylinder and the volume recorded. The cylinder is then tapped against a surface a specified number of times and the volume again recorded. The poured and tapped densities are then determined and the Elausner Ratio=tapped density/poured density calculated. A ratio of  less than 1.25 indicates a free flowing material while a ratio of  greater than 1.5 indicates a poor flowing (cohesive) material. Another possible method involves the measurement the Angle of Repose by pouring material through a funnel held at a fixed height onto a piece of graph paper until a cone is formed. The height (H) and the radius (R) of the cone is determined and the angle calculated (tan xcex8=H/R). An Angle of Repose xcex8 less than 300 indicates good flow properties while an Angle of Repose xcex8 greater than 400 indicates very poor flow properties (James I. Wells, Pharmaceutical Preformulation, Ellis Horwood Series in Pharmaceutical Technology, 1988).
The size of the microparticles, which includes microcapsules and especially microspheres, is preferably in the range 1 to 200 xcexcm, more preferably 1 to 100 xcexcm, as measured by e.g. light microscopy or sieve fractionation.
The microparticles will consist of preferably between 50 and 95%, more preferably between 70 and 90% and most preferably between 75 and 85% of type A gelatin, and correspondingly between 50 and 5%, preferably between 30 and 10% and most preferably between 25 and 15% of chitosan, as measured in relation to the total amount of gelatin and chitosan in the final composition (i.e. excluding therapeutic agent and other ingredients which may be included).
The term xe2x80x9ctherapeutic agentxe2x80x9d includes drugs, genes (DNA) or gene constructs, vaccines and components thereof (for example isolated antigens or parts thereof) and monoclonal antibodies. For applications employing such materials as genes, gene constructs, vaccines and monoclonal antibodies, the microparticles can be used to enhance the delivery of the therapeutic agent into the mucosal tissue for enhanced therapeutic effect, for example presentation of an antigen to the underlying lymphoid tissue, and/or transfection of the cells in the mucosal lining.
Preferably the therapeutic agent is a polar drug. By xe2x80x9cpolar drugsxe2x80x9d we means molecules with a partition coefficient (octanolxe2x80x94water system) of less than 50.
The compositions may be used with therapeutic agents selected from the following non-exclusive list: insulin, PTH (parathyroid hormone), PTH analogues, PTHrP (human parathyroid hormone peptide), calcitonins (for example porcine, human, salmon, chicken or eel) and synthetic modifications thereof, enkephalins, LHRH (luteinising hormone releasing hormone) and analogues (nafarelin, buserelin, leuprolide, goserelin), glucagon, TRH (thyrotropine releasing hormone), vasopressin, desmopressin, growth hormone, heparins, GHRH (growth hormone releasing hormone), CCK (cholecystokinin), THF (thymic humoral factor), CGRP (calcitonin gene related peptide), atrial natriuretic peptide, nifedipine, metoclopramide, ergotamine, pizotizin, pentamidine and vaccines (particularly but not limited to AIDS vaccines, measles vaccines, rhinovirus Type 13 and respiratory syncytial virus vaccines, influenza vaccines, pertussis vaccines, meningococcal vaccines, tetanus vaccines, diphtheria vaccines, cholera vaccines and DNA vaccines (e.g. one containing a plasmid DNA coding for a suitable antigen)).
Further therapeutic agents include but are not limited to: antibiotics and antimicrobial agents, such as tetracycline hydrochloride, leucomycin, penicillin, penicillin derivatives, erythromycin, sulphathiazole and nitrofurazone; anti-migraine compounds, such as naratriptan, sumatriptan, alnitidan or other 5-HT1 agonists; vasoconstrictors, such as phenylephedrine hydrochloride, tetrahydrozoline hydrochloride, naphazolinie nitrate, oxymetazoline hydrochloride and tramazoline hydrochloride; cardiotonics, such as digitalis and digoxin; vasodilators, such as nitroglycerine and papaverine hydrochloride; bone metabolism controlling, agents, such as vitamin D and active vitamin D3; sex hormones; hypotensives; anti-tumour agents; steroidal anti-inflammatory agents, such as hydrocortisone, prednisone, fluticasone, prednisolone, triamcinolone, triamcinolone acetonide, dexamethasone, betamethasone, beclomethasone and beclomethasone dipropionate; non-steroidal anti-inflammatory agents, such as acetaminophen, aspirin, aminopyrine, phenylbutazone, mefanic acid, ibuprofen, diclofenac sodium, indomethacin, colchicine and probenecid; enzymatic anti-inflammatory agents, such as chymotrypsin and bromelain seratiopeptidase; anti-histaminic agents, such as dephenhydramine hydrochloride, chloropheniramine maleate and clemastine; anti-tussive-expectorants, such as codeine phosphate and isoproterenol hydrochloride; analgesics, such as opioids (like diamorphine, morphine and its polar metabolites, such as morphine-6-glucuronides and morphine-3-sulphate); anti-emetics, such as metoclopramide, ondansetron, chlorpromazine; drugs for treatment of epilepsy, such as clonazepam; drugs for treatment of sleeping disorders, such as melatonin; drugs for treatment of asthma, such as salbutamol.
Combinations of the abovementioned therapeutic agents may be employed.
The compositions according to the invention may be administered orally, nasally, vaginally, buccally, rectally, via the eye, or via the pulmonary route, in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art. For example, compositions may be administered via the nasal route as a powder using a nasal powder device, via the pulmonary route using a powder inhaler or metered dose inhaler, via the vaginal route as a powder using a powder device, formulated into a vagina suppository or pessary or vaginal tablet or vaginal gel, via the buccal route formulated into a tablet or a buccal patch, via the rectal route formulated into suppositories; via the eye in the form of a powder or a dry ointment, and via the oral route in the form of a tablet, a capsule or a pellet (which compositions may administer agent via the stomach, the small intestine or the colon), all of which may be formulated in accordance with techniques which are well known to those skilled in the art. The compositions may gel on the mucosa at least to some extent and this may facilitate retention of the composition on the mucosa.
The preferred route of administration is nasal. Devices which may be used to deliver the compositions according to the invention nasally include the Direct Haler(copyright), the Bespak(copyright) powder device, the Monopoudre(copyright) (Valois) and the Insufflator(copyright) (Teijin).
Compositions according to the invention which may be administered orally may be adapted to deliver therapeutic agent to the small intestine or the colonic, especially the proximal colonic, region of the gastrointestinal tract.
Preferably, a means is provided to prevent release of therapeutic agent until the formulation reaches the small intestine or colon. Means which may be employed in order to prevent release until the small intestine is reached are well known to those skilled in the art (see for example dosage forms coated with so-called enteric polymers that do not dissolve in the acidic conditions which exist in the stomach, but dissolve in the more alkaline conditions found in the small intestine of a mammal. Suitable enteric coating materials include modified cellulose polymers and acrylic polymers and in particular those sold under the trademark Eudragit(copyright).) Means which may be employed in order to prevent release until the colon is reached are well known to those skilled in the art. Such materials include cellulose acetate trimellitate (CAT), hydroxypropylmethyl cellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP) and shellac, as described by Healy in his article xe2x80x9cEnteric Coatings and Delayed Releasexe2x80x9d, Chapter 7 in Drug Delivery to the Gastrointestinal Tract, eds. Hardy et al, Ellis Horwood, Chichester, 1989). Especially preferred materials are methylmethacrylates or copolymers of methacrylic acid and methylmethacrylate. Such materials are available as Eudragit(copyright) enteric polymers (Rohm Pharma, Darmstadt, Germany). Such a coating may also suitably comprise a material which is redox-sensitive (e.g. azopolymers which may, for example, consist of a random copolymer of styrene and hydroxyetlyl methacrylate, cross-linked with divinylazobenzene synthesised by free radical polymerisation, or disulphide polymers (see PCT/BE91/00006 and Van den Mooter, Int. J. Pharm. 87, 37(1992)). See also International Patent Application WO 97/05903.
It will be appreciated by those skilled in the art that the site of delivery may also be selectively controlled by varying the thickness of certain of the abovementioned polymer coatings.
It will be well understood by those skilled in the art that further excipients may be employed in formulations comprising the compositions according to the invention. For example, in solid dosing forms, further excipients which may be employed include diluents such as microcrystalline cellulose (e.g. Avicel(copyright), FMC), lactose, dicalcium phosphate and starch(es); disintegrants such as microcrystalline cellulose, starch(es) and cross-linked carboxymethylcellulose; lubricants such as magnesium stearatc and stearic acid; granulating agents such as povidone; and release modifiers such as hydroxypropyl methylcellulose and hydroxypropyl cellulose. Suitable quantities of such excipients will depend upon the identity of the active ingredient(s) and the particular dosing form which is used.
If desired, other materials may be included in the composition, for example absorption enhancers. Suitable absorption enhancers include non-ionic surfactants, cyclodextrins, bile salts and, preferably, phospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol and generally those mentioned in WO 88/09163.
According to a further aspect of the invention, there is provided a pharmaceutical formulation in a form suitable for administration to a mucosal surface which comprises a composition according to the invention in a pharmaceutically acceptable dosage form.
Compositions according to the invention have been found to have the advantage that they provide improved transport of polar drugs across mucosal surfaces, such as the nasal cavity, have improved flow properties when compared to prior art compositions, and avoid the need for the use of chemical crosslinking agents.
According to a further aspect of the invention there is thus provided a method for the improved transport of therapeutic agents across (or into) mucosal surfaces (which includes the presentation of vaccines to mucosal surfaces) in mammals, and a method of treating a human or other mammal, which methods comprise administering a composition, as described above, preferably to a mucosal surface of that human or other mammal, for example the vagina, buccal cavity, rectum, lungs, eye, colon, small intestine, stomach or nasal cavity.
The amount of therapeutic agent which may be employed in the compositions according to the invention will depend upon the agent which is used. However, it will be clear to the skilled person that suitable doses of therapeutic agents can be readily determined non-inventively. Suitable doses are in the range 1 xcexcg to 1 g depending upon the therapeutic agent(s) which is/are employed and the route of administration.